gms | German Medical Science

Objectives: Implant-associated infections with S. aureus represent a serious complication associated with revision surgery, increased morbidity and increased costs. Besides biofilm formation, invasion of osteoblasts by S. aureus might play an important role in implant-associated infection, since the bacteria can evade the immune response and antibiotic therapy. First step here is the adhesion towards the eukaryotic cell. Here we analyse factors involved in adherence on the surface of osteoblast like cells.

Methods: S. aureus ATCC® 29213 as well as 8 isolates from patients with an implant-associated infection were used in this study. Isolates were characterised with respect to MSCAMMs by PCR and mass spectroscopy. Adhesion on and invasion in osteoblast like cell lines SaOS2 and MG63 was determined. Bacteria were cultured in LB-medium with the addition of glucose, NaCl and their combinations.After reaching logarithmic or stationary growth phase bacteria were harvested, washed in PBS and added on confluent cell layers of the osteoblast like cells lines. After incubation for 30 minutes, non-adherent S. aureus were washed off. The remaining bacteria were detached by lyses of osteoblast like cells and plated on LB-agar, in order to perform cell count. Invasion of S. aureus into the cells was determined after 1 to 3 days of cultivation in presence of antibiotics. The expression level of known genes associated with adhesion (MSCRAMMs) were determined with qPCR.

Results and conclusion: Adhesion of S. aureus ATCC 29213 on osteoblasts like cells increased when cultured to the stationary growth phase and decreased when cultured under biofilm conditions. Here gene expression of MSCRAMMs correlated with adhesion. There was a significant difference in S. aureus ATCC 29213 adhesion to the two osteoblast cell lines, indicating the possibility of two different mechanisms. Focussing on the clinical isolates, 3 of 8 adhered to the osteoblast cell lines, here without difference between the cell-lines used.

With respect to bone, genes for several proteins associated with adhesion like cna, bbp, fnbB could not be detected in adhesive isolated. On the other side fnbA, could be detected in all isolates, without significant differences in expression. Although it is common sense that the absence of fnbA leads to reduced adhesion, the presence of either the gene or the protein does not guarantee adhesion to osteoblasts, making the role of fnbA questionable.

Furtherly, qPCR analyses showed that clfA might be a candidate gene because of the expression pattern in the S. aureus isolates.

With respect to invasion into the osteoblast like cells, as a consequence of adhesion, we found differences between SaOS2 and MG63 with respect to survival of the bacterial isolates. Currently proteome analysis is performed in order to detect the factors involved in internalisation and survival.